Method of inhibiting secretory effects caused by cholera

ABSTRACT

The invention relates to the treatment fluid secretion caused by Cholera toxin that can be blocked through the administration of PAF antagonists BN 52021 and SR 27417.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 08/040,444 filed on Apr. 1,1993, now U.S. Pat. No. 5,436,239, which is a CIP of Ser. No. 07/861,620 filed Apr. 1, 1992, abandoned.

FIELD OF THE INVENTION

The invention relates to the treatment of antibiotic associated colitis, typically due to Clostridium difficile using Platelet Activating Factor antagonists, such as WEB 2170, SR 27417 or BN 52021, or the cyclooxygenase antagonists, such as indomethacin. The PAF antagonists BN 52021 and SR 27417 and the cyclooxygenase antagonist indomethacin were effective in inhibiting the secretory effects caused by C. difficle Toxin A and even by Cholera toxin.

BACKGROUND OF THE INVENTION

Clostridium difficile is one of the most frequently recognized bacterial causes of diarrheal disease in hospitalized adults in industrialized countries. The microorganism can be acquired nosocomially and is present in environmental sources. Antibiotic associated colitis and pseudomembranous colitis are frequently associated with cytotoxigenic Clostridium difficile. The frequency of Clostridium difficile toxin associated with antibiotic associated colitis is 50-80% and with pseudomembranous colitis is 90-100%. Despite available treatment for antibiotic associated colitis and pseudomembranous colitis, relapses occur in 20-25% of patients. Vancomycin and metronidazole can be effective, but are subject to relapse after the use of the drugs may occur.

Clostridium difficile produces two toxins, A (enterotoxic) and B (cytopathic), the former (Toxin A) being implicated in the pathogenesis of pseudomembranous colitis. Toxin A causes hemorrhagic fluid accumulation associated with mucosal damage and a cytopathic effect in tissue culture cells. In experimental animals such as rabbits, toxin A causes hemorrhagic fluid secretion and cell damage in ligated intestinal segments of loops, and is considered to be the cause of antibiotic-associated colitis in experimental hamsters and in people.

Toxin B is a more potent cytotoxin than toxin A, but it has no enterotoxic activity. It has been reported that the toxin A is important in the pathogenesis of Clostridium difficile disease. In animal models of the disease, Clostridium difficile culture supernatant filtrate or purified toxin A causes mucosal lesions characteristic of antibiotic-associated colitis. In human disease, as well as in animal models, a spectrum of mucosal changes ranging from nonspecific colitis (with or without diarrhea) to severe disease with complete mucosal necrosis has been described in prior literature.

Antibiotics, including vancomycin or metronidazole, directed against C. difficile, or anion exchange resins that bind the toxins of C. difficile, including cholestyramine, have been used to treat C. difficile diarrhea. However, the antibiotics prolong carriage of C. difficile and relapses are common and resins are not always well tolerated.

PAF inhibitors have been have been found to be effective in treating Inflammatory Bowel Disease (IBD) when administered 4-7 days after the induction of colitis. The PAF inhibitor BN52021, however, shows no effect in the first four days. Chemical Abstracts 110:190302k (1989). IBD is distinct from the specific Clostridium difficile toxin-induced antibiotic associated acute colitis. IBD, including Crohn's disease, is a chronic, recurring, granulomatous inflammatory process that is largely related to host susceptibility. C. difficile diarrhea is a specific, acute, toxinsmediated diarrhea that can occur in any individual and does not recur over many years as is characteristic of IBD. Clostridium difficile is defined as "a species that is part of the normal colon flora in human infants and sometimes in adults. It sometimes produces a toxin that causes pseudomembranous enterocolitis in patients receiving antibiotic therapy" Dorland's Illustrated Medical Dictionary, W. B. Saunders Co., Philadelphia, Pa., 27th Edition, 1988, p. 348. Pseudomembranous enterocolitis is defined as "an acute inflammation of the bowel muscosa with the formation of pseudomembranous plaques overlying an area of superficial ulceration, and the passage of the pseudomembranous material in the feces; it may result from shock and ischemia or be associated with antibiotic therapy. Dorland's Illustrated Medical Dictionary, supra, p. 560. Crohn's disease is defined as "a chronic granulomatous inflammatory disease of unknown etiology, involving any art of the gastrointestinal tract from mouth to anus, but commonly involving the terminal ileum with scarring and thickening of the bowel wall; it frequently leads to intestinal obstruction and fistula and abscess formation and has a high rate of recurrence after treatment." Dorland's Illustrated Medical Dictionary, supra, p. 484. IBD is, to an extent, susceptible to control by diet, fitness and stress reduction. Eating Right for a Bad Gut: The Complete Nutritional Guide to Ileitis, Colitis, Crohn's Disease & Inflammatory Bowel Disease, Dr. James Scala, Penguin.

SUMMARY OF THE INVENTION

It has now been found that the hemorrhagic fluid secretion caused by Clostridium difficile toxin A in ligated intestinal segments can be significantly blocked by administering an effective amount of either a Platelet Activating Factor (PAF) inhibitor, or by a cyclooxygenase inhibitor, or by the combination of inhibitors. The PAF inhibitors are from the group consisting of BN52021, WEB 2170 or SR27417. The phospholipase A₂ inhibitor is from the group consisting of aristolochic acid, bromophenacyl bromide and quinacrine. The cyclooxygenase inhibitor is indomethacin.

The fluid secretion caused by Cholera toxin in ligated intestinal segments can be blocked by administering an effective amount of a Platelet Activating Factor inhibitor selected from the group consisting of BN52021 and SR27417 or the cyclooxygenase inhibitor indomethacin. The combination of the cyclooxygenase inhibitor indomethacin and at least one Platelet Activating Factor antagonist from the group consisting of SR 27417 and BN 52021 also blocks the hemorrhagic fluid secretion caused by Cholera toxin.

BRIEF DESCRIPTION OF THE DRAWINGS

The specification and disclosed invention will be better understood when read in reference to the drawings in which:

FIG. 1 is a graph illustrating the effects of Aristolochic Acid on C. difficile toxin A in rabbit ileum;

FIG. 2 is a chart illustrating the effects of Aristolochic Acid on C. difficile toxin A in rabbit ileal ligated loops;

FIG. 3 is a graph illustrating the effects of PAF inhibitors (BN, WEB, SR) and indomethacin on C. difficile toxin A in rabbit ileum;

FIG. 4 is a chart illustrating the effects of PAF inhibitors (BN, WEB, SR) or indomethacin on C. difficile toxin A in ligated rabbit ileal loops;

FIG. 5 is a graph illustrating the effects of Quinacrine on C. difficile toxin A in rabbit ileum;

FIG. 6 is a chart illustrating the effects of Quinacrine on C. difficile toxin A in ligated rabbit ileal loops;

FIG. 7 is a chart illustrating the effects of Quinacrine on C. difficile toxin A in T84 cell monolayers;

FIG. 8 is a graph illustrating the effects of Quinacrine on C. difficile toxin A in T84 cell monolayers;

FIG. 9 is a graph illustrating the effects of PAF inhibitors (BN and SR) and indomethacin on cholera toxin in rabbit loops; and,

FIG. 10 is a graph illustrating the effects of bromophenacyl bromide on C. difficile toxin A in rabbit ileum.

DETAIL DESCRIPTION OF THE PREFERRED EMBODIMENTS

The hemorrhagic fluid secretion caused by Clostridium difficile toxin A in ligated intestinal segments can be significantly blocked by either Platelet Activating Factor (PAF) inhibitors BN52021, WEB 2170, SR27417; phospholipase A₂ inhibitors, Aristolochic Acid, Quinacrine, and bromophenacyl bromide or by cyclooxygenase inhibitor indomethacin, or by the combination of BN 52021 or WEB 2170 and indomethacin.

CRC Handbook of PAF and PAF Antagonists, Edited by Braquet, CRC Press, Boston Mass., identifies WEB 2170 on page 51. The ginkgolide BN 52021 (IHB nomemclature) is illustrated on page 52 of the CRC Handbook, the subject matter of the CRC Handbook being incorporated herein by reference as though completely reproduced.

The structural formula for BN 52021, with R¹ and R² equal OH and R³ equals H, is as follows: ##STR1##

The structural formula for WEB 2170 is as follows: ##STR2##

TIme availability of highly purified Clostridium difficlle toxin A, has allowed accurate, reproducable study of the specific effects involved in early mucosal damage end its correlation with the intestinal response. The preparation of toxin A and the use of toxin A in studies in ligated small intestinal segments and other studies, is disclosed in Laboratory Investigation, Vol. 61, No. 4, 1989, pages 419 through 425, Clostridium difficile Toxin A, Lima et al., the subject matter of which is incorporated herein by reference, as though completely reproduced.

The instant disclosure conducted tests using: (1) platelet activating factor (PAF) inhibitors, BN52021 (BN), WEB2170 (WEB) and SR27417 (SR); (2) the phospholipase A₂ inhibitors, arisrtolochic acid (AA), Quinacrine (QN) and bromophenacyl bromide (BPB); and (3) cyclooxygenase inhibitor, indomethacin (INDO). These were tested both in vivo in the rabbit ileal ligated loop model and in vitro in monolayers of T-84 cells in tissue culture (a colonic epithelial carcinoma cell line) to determine the effects of these inhibitors on the cytotoxicity and secretory responses due to C. difficile toxin A.

EXPERIMENT I

Clostridium difficile toxin A alone and in combination with the inhibitors were placed in 4-6 cm ligated Ileal segments in 1.5-2.5 kg, male, New Zealand white rabbits. The rabbits were fasted overnight and provided water ad libitum. The animals were anesthetized with an initial intramuscular dose of ketamine (35 mg/kg body weight) and xylazine (5 mg/kg). Ligated rabbit small intestinal segments of approximately 5 cm were made with double ties of umbilical tape. These segments were injected with the following combinations and then returned to the abdominal cavity without separation from the remaining intestine or interruption of the blood supply. After six (6) hours the animals were sacrificed and the fluid volume (milliliters) and length (centimeters) for each loop was measured and a volume to length ratio calculated. Toxin A 10 μg/segment in 1 ml consistently caused severe hemorrhagic inflammatory fluid secretion at 6-8 hours (V/L=0.64±0.13 ml/cm segment, n=6). The molarity (M) indicates the drug concentration in the 1 ml containing the 10 μg/ml Toxin A. Phosphate buffered saline (PBS) is used as a control.

Each rabbit was injected with one of the following:

1 ml toxin A in PBS (10 μg/ml)

1 ml PBS (as a control),

1 ml toxin A and BN52021 (10⁻⁴ M and 10⁻⁵) [BN]

1 ml toxin A and WEB2170 (10⁻⁵ M and 10⁻⁶ M) [WEB]

1 ml toxin A and SR27417 (10⁻⁵ and 10⁻⁶ M) [SR]

1 ml toxin A and Aristolochic acid (10⁻⁵ M) [AA]

1 ml toxin A and a combination of quinacrine (10⁻⁵ M) [QN], indomethacin (10⁻⁵ M) [INDO]

1 ml toxin A and bromophenacyl bromide(10⁻⁵) [BPB]

0.5 ml toxin A (20 μg/ml) plus 0.5 ml of BN(2×10⁻⁴ M and 2×10⁻⁵)

0.5 ml toxin A and WEB(2×10⁻⁵ M and 2×10⁻⁶)

0.5 ml toxin A and SR(2×10⁻⁵ M and 2×10⁻⁶)

0.5 ml toxin A and AA(2×10⁻⁵ M)

0.5 ml toxin A and QN(2×10⁻⁵ M)

0.5 ml toxin A and INDO(2×10⁻⁵ M)

The toxin A 10 μg/segment hemorrhagic inflammatory fluid secretion was significantly inhibited by 10⁻⁵ M AA, a phospholipase A₂ inhibitor. AA provided an 87% inhibition, (p<0.0001, n=6). The secretory effect of toxin A was also inhibited in ligated segments adjacent to those with AA (89% inhibition, p≦0.0001, n=6). The results of these tests are illustrated in the graph of FIG. 1 and the chart of FIG. 2.

The PAF inhibitors, BN (10⁻⁴ M and 10⁻⁵ M), WEB (≧10⁶ M) or the cyclooxygenase inhibitor, INDO (10⁵ M), both significantly inhibited the secretory effects of toxin A in ligated but not adjacent rabbit intestinal segments (71% inhibition for BN; 84% for WEB; 79% for INDO; all p<0.0001) as noted in FIGS. 3 and 4. illustrate the test results using p factors of <0.001 and <0. SR gave a reduction of 49% (p<0.001) in the same loops and a reduction of 48% (p<0.01) in the adjacent loops.

The secretory effects of C. difficile toxin A with ligated ileal segments were significantly inhibited by phospholipase A₂ inhibitor, QN (p<0.05), in both the same or the adjacent loops with toxin A. The test results where p<0.015 and p<0.008 are illustrated in FIGS. 5 and 6.

The phospholipase A₂ pathway is involved in the secretory responses to C. difficile toxin A, via the subsequent generation of prostaglandin and/or PAF. The prostaglandin is decreased by the phospholipase The generation of prostaglandin is reduced by the PLA₂ or cyclooxygenase inhibitors.

The effects of BPB on the D. difficile toxin A are illustrated in FIG. 10, where p<0.01 end n=4.

The numeric results of the experiments with BN, INDO and WEB are illustrated in Table I.

                                      TABLE 1                                      __________________________________________________________________________     Results for Studies of Inhibition of C. diff Toxin A induced Secretion         in Rabbits' Loops by PAF and Cyclooxygenase Inhibitors                         (Vol/Length ratios, μl/cm)                                                                   BN vs Toxin                                                                            INDO                                                                               WEB vs Toxin                                      Rabbit   Toxin                                                                              PBS 10.sup.-4                                                                          10.sup.-5                                                                          10.sup.-5                                                                          10.sup.-5                                                                          10.sup.-6                                     __________________________________________________________________________     1 (2.1 kg)                                                                              43.5                                                                               20.0                                                                       104.2                                                                              8.8                                                               2 (2.0 kg)                                                                              81.4                                                                               12.5                                                                               58.3    19.6                                                           175.0                                                                              0.0 106.4   0.0                                                   1 (2.0 kg)                                                                              175.9                                                                              20.8                                                                       186.1                                                                              15.6                                                                       114.8                                                                 2 (2.2 kg)                                                                              277.8                                                                              159.1                                                                              59.5                                                                               127.5                                                                              69.8                                                           172.8                                                                              55.6                                                                               42.6                                                                               59.5                                                                               61.0                                                  1 (1.6 kg)                                                                              111.1                                                                              22.2    57.1                                                               142.9                                                                              50.0    66.7                                                               228.6                                                                          233.3                                                                 2 (2.1 kg)                                                                              184.6                                                                              66.7            60.0                                                                               54.5                                                   300.0                                                                              25.0            40.0                                                                               40.0                                          1 (2.0 kg)                                                                              140.0                                                                              10.0                                                                       150.0                                                                              9.1                                                               2 (2.1 kg)                                                                              276.6                                                                              9.1 55.6                                                                               60.0                                                               342.9                                                                              21.3                                                                               72.7                                                                               80.0                                                      3 (2.2 kg)                                                                              280.0                                                                              40.0            20.0                                                                               107.7                                                  180.0                                                                              8.3             33.3                                                                               83.3                                          Mean     185.8                                                                              30.8                                                                               65.9                                                                               75.1                                                                               37.6                                                                               38.3                                                                               71.4                                          SD       77.9                                                                               36.9                                                                               22.1                                                                               27.0                                                                               33.3                                                                               16.7                                                                               36.9                                          n        21  18  6   6   4   4   4                                             % inhibition     77.4                                                                               71.4                                                                               95.6                                                                               95.2                                                                               73.8                                          p            <0.001                                                                             0.001                                                                              0.002                                                                              0.001                                                                              0.001                                                                              0.009                                         __________________________________________________________________________     Final comparation                                                                         Toxin                                                                               PBS  BN   WEB  INDO                                            __________________________________________________________________________     Mean       185.8                                                                               30.8 70.5 62.5 37.6                                            SD         77.9 36.9 24.0 31.2 33.3                                            % inhibition         74.4 79.5 95.6                                            p                    <0.001                                                                              <0.001                                                                              0.001                                           __________________________________________________________________________

The hemorrhagic fluid secretion caused by Clostridium difficile is significantly blocked by two Platelet Activating Factor (PAF) inhibitions, BN 52021 (10⁴ M) or WEB 2170 (≦10⁶ M) or by the cyclooxygenase inhibitor indomethacin (10⁻⁵ M); inhibition was 62% with BN52021, 74% for WEB 2170, 65% for indomethacin, all p<0.05). PAF inhibitors, alone or in combination with cyclooxygenase inhibitors, provide a means to block the common and often severe antibiotic-associated diarrhea and colitis caused by Clostridium difficile.

EXPERIMENT II

T84 cells, a human intestinal epithelial cell line, were grown, passaged, then grown on the 4-well LAB-TEK tissue culture chamber slides (Nunc, Inc., Naperville, Ill.) to confluency. C. difficile toxin A 0.7 μg/well (1 ml, final concentration 0.7 μg/ml) alone was placed onto a T84 cell monolayer. Additional cell monolayers were applied with equal concentration of toxin A in combination with:

1 ml BN(10⁻⁴ M),

1 ml WEB(10⁻⁴ M)

1 ml AA(5×10⁻⁴ M)

1 ml QN(5×10⁻⁴ M)

1 ml BPB(10⁻⁴ M)

1 ml INDO(10⁻⁴ M)

Cell monolayers were prepared with toxin A 0.7 μg (0.5 ml of 1.4 μg/ml). Additional cells were prepared with the same toxin A concentration in combination with:

0.5 ml BN(2×10⁻⁴ M)

0.5 ml WEB(2×10⁻⁴ M) or

0.5 ml AA(10⁻⁴ M)

0.5 ml QN(10⁻³ M)

0.5 ml BPB(2×10⁻⁴ M)

0.5 ml INDO(2×10⁻⁴ M)

After eight (8) hours incubation, the cell-coated chamber slides were rinsed in PBS and fixed for 15 minutes at room temperature in 3.7% formaldehyde. The slides were rinsed again in PBS, permeabilized for five (5) minutes in 20° C. acetone, air dried, stained with rhodamine-labeled phalloidin for 30 minutes at room temperature in the dark, rinsed twice in PBS and kept in the dark at 4° C. Cells were then examined and photographed using an inverted Olympus fluorescence microscope. To compare the pattern of labeling between toxin-treated and PAF inhibitors or phospholipase A₂ inhibitors or cyclooxygenase inhibitor plus toxin-treated mono-layers, photomicrographs were taken with Kodak 400, daylight slides and exposures were varied to optimize the resulting images.

The destruction by toxin A (0.7 μg/ml) of rhodamine-labeled phalloidin-stained F-actin at the tight junctions of T-84 cell monolayers was inhibited by 5×10⁻⁴ M AA or 10⁻⁴ M BPB (n=2-4 experiments each). BN, INDO, WEB, and QN also inhibited the reduction in F-acting staining by toxin A.

EXPERIMENT III

T84 cells, a human intestinal epithelial cell line, were grown, passaged and mounted on collagen-coated microfilters. Transepithelial resistances generated by the ring-mounted monolayers used for these studies ranged from 2600 to 4000Ω cm².

When the T84 cells grown on the microfilters reached confluence as determined by phase microscopy and development of a high, stable resistance, toxin A 0.02 μg(10 μl, final concentration 0.07 μg/ml), or 36 μl QN(2×10⁻⁴ M, final concentration 2×10⁻⁵ M), or toxin A 0.02 μg plus 36 μl QN(2×10⁻⁴ M) were added into 300 μl tissue culture medium. All above reagents were applied only to the apical (mucosal) surface of the monolayers. Resistance was measured every hour during the first eight hours and then at 10, 12 and 24 hours.

The results of the QN resistance are shown in FIGS. 7 and 8. Simultaneous administration of QN (p<0.05) additionally significantly inhibited tissue resistance in the T84 cell monolayers caused by toxin A at 5-6 hours exposure.

The foregoing PAF inhibitors, BN and SR and Indomethacin, have been found to significantly reduce the enterotoxin Cholera toxin (choleragen) produced by Vibrio cholerae, as well as C. difficile. Choleragen acts on the epithelial cells in the small intestine to cause secretion of large quantities of isotonic fluid from the mucosal surface. Choleragen is affected by the PAF inhibitors similarly to the toxin A created by Clostridium difficile. The Cholera related diarrhea results in massive gastrointestinal fluid loss and saline depletion, acidosis and shock. The ability to control the effects of the diarrhea would allow the body to absorb badly needed fluids, nutrients and medication, and eliminate the further depletion of the body's resources and resulting deficiencies.

EXPERIMENT IV

Cholera toxin, alone and in combination with the inhibitors, BN, SR and indomethacin, were placed in 4-6 cm ligated Ileal segments in 1.5-2.5 kg, male, New Zealand white rabbits. The rabbits were fasted overnight and provided water ad libitum. The animals were anesthetized with an initial intramuscular dose of ketamine (35 mg/kg body weight) and xylazine (5 mg/kg). Ligated rabbit small intestinal segments of approximately 5 cm were made with double ties of umbilical tape. These segments were injected with the following combinations and then returned to the abdominal cavity without separation from the remaining intestine or interruption of the blood supply. After six (6) hours the animals were sacrificed and the fluid volume (milliliters) and length (centimeters) for each loop was measured and a volume to length ratio calculated. Choleragen 10 μg/segment in 1 ml consistently caused severe hemorrhagic inflammatory fluid secretion at 6-8 hours (V/L=0.64±0.13 ml/cm segment, n=6). The molarity (M) indicates the drug concentration in the 1 ml containing the 10 μg/ml choleragen.

Each rabbit was injected with one of the following:

1 ml choleragen in PBS (1 μg/ml)

1 ml PBS (as a control),

1 ml choleragen and BN52021 (⁻⁴ M and 10⁻⁵ M) [BN]

1 ml choleragen and SR27417 (10⁻⁵ and 10⁻⁶ M) [SR]

1 ml choleragen and a combination of BN52021 (10⁻⁵ M) [BN], indomethacin (10⁻⁵ M) [INDO]

0.5 ml choleragen and SR(2×10⁻⁵ M and 2×10⁻⁶)

0.5 ml choleragen and BN(2×10⁻⁵ M)

0.5 ml choleragen and INDO(2×10⁻⁵ M)

The choleragen 1 μg/segment hemorrhagic inflammatory fluid secretion was significantly inhibited by 10₅ -10⁶ M SR27417. SR provided an 86% inhibition when placed in the loop with the toxin, as well as a slightly lesser inhibition when in the loop adjacent to the toxin, (both p<0.011), as illustrated in FIG. 9.

The PAF inhibitors, BN (10⁻⁴ M and 10⁻⁵ M), and INDO (10⁵ M), both significantly inhibited the secretory effects of toxin A in ligated intestinal segments (44% inhibition for BN; 38% for INDO, both p<0.01). 

What is claimed is:
 1. The method of blocking hemmorhagic fluid secretion caused by Cholera toxin in the gastro-intestinal tract, comprising the administration of an effective amount of an inhibitor to reduce said fluid secretion.
 2. The method of claim 1 wherein said inhibitor is an inhibitor of Platelet Activating Factor action selected from the group consisting of BN52021 and SR27417.
 3. The method of claim 2 wherein said inhibitor of Platelet Activating Factor action is administered by injection.
 4. The method of claim 2 wherein said inhibitor of Platelet Activating Factor action is a tablet. 